The present invention is in the field of biotechnology and is related to a new antibiotic inducible/repressible genetic construct or system for improving particle the control of gene therapy or gene immunization. In particular, a system or genetic construct adapted for all types of gene therapy and gene immunization based upon the use of naked DNA or DNA incorporated into various vectors (such as plasmide, adeno-associated viruses, autonomous parvoviruses, retroviruses or adenoviruses or a combination thereof).
Various systems comprising naked DNA or DNA incorporated into a suitable vector (plasmid, virus, cationic visicule, . . . ) are used in gene therapy or gene immunization (vaccine). Various publications describe for gene transfer into cells, the use of adeno-associated viruses, which are human defective parvoviruses whose genomes are made of single stranded DNA molecules. Six or five different serotypes have been cloned in prokaryotic plasmide and could be used to derive vectors.
The international patent application PCT/US95/04587 describes gene delivery to adult CNS using AAV vectors.
Humans suffering from Parkinsonism have been treated by striatal implantation of foetal dopaminergic neurons (Lindvall et al., Arch. Neurol. 46:615-631 (1989); Widner et al. New Engl. J. Med. 327: 1556-1563 (1992). Following surgery, the patients exhibited improvement of neurological function. Grafts partially re-establish dopaminergic activity and ameliorate motor functions. However, the success of foetal brain tissue transplantation into impaired area of Parkinson""s and Huntington""s patients brain is limited by the poor survival of the graft. To ensure maximal viability, the foetal tissue must be freshly harvested prior to transplantation. Recent advances consist of keeping the tissue refrigerated (at 4xc2x0 C.) for 24 hours without loss of viability. Nevertheless, the coordination between the harvesting of the foetal tissue and the transplantation procedure is still a problem. Furthermore, the amount of foetal tissue available for transplantation is limited for practical and ethical reasons. Foetal tissue is technically difficult to obtain, particularly if multiple donors are needed for each patient. This limits the widespread applicability of foetal tissue transplantation.
The supply of Glial cell line-Derived Neurotrophic Factor (GDNF), a neurotrophic factor for dopaminergic neurons, could promote the protection of rafted cells as well as of remaining host dopaminergic cells. However, since neurotrophic factors can not cross he brain-blood-barrier, they have to be administrated directly in the brain in sustained levels.
The international patent application (PCT)/US96/05814 describes a method of using neurotrophic actors to enhance neuronal survival and promote functional integration of grafted neurons using osmotic umps implanted in the brain. This technique is difficult to implement in the clinics, in particular because of the risk of bacterial contamination.
Improving the survival of the grafted tissue by transfer of genes coding for neurotrophic factors would reduce the amount of tissue needed per patient and make the transplantation therapy available to a greater number of patients.
Stable genetic modification of the graft cells by the means of viral vectors expressing trophic factors could be used to enhance the survival of the grafted tissue.
Genetically-modified foetal mesencephalon fragments or dissociated cell suspensions expressing GDNF could be grafted in order to obtain i) a better survival of the graft (autocrine effect), ii) the protection of host""s dopaminergic terminals in the putamen and of a dopaminergic cell bodies in the substantia nigra after retrograde transport of GDNF (paracrine effect).
Furthermore, the combination of autocrine and paracrine effects could result in a better correction of parkinsionnian symptoms by foetal grafts transplants.
Adeno-associated virus is a human defective parvovirus whose genome is a single stranded DNA molecule. Five different serotypes have been cloned in prokaryotic plasmide and could be used to derive vectors.
For efficient replication AAV requires a co-infection with a so-called xe2x80x9chelper virusxe2x80x9d, usually adenovirus or herpes simplex virus. In the absence of helper virus, AAv can still enter host cells but it stays latent with his genome integrated in the cellular genome. The genome is flanked by 2 inverted terminal repeats (ITRs) which serve as a replication origin.
The double-stranded form of AAV type 2 has been cloned in a pBR322 plasmid allowing the genetic analysis of the virus as well as the development of vectors for gene transfer (Samulski et al. 1982).
It was soon realized the non-coding ITRs are the only elements required in cis for replication and encapsidation of the viral genome (McLaughlin et al., 1987). Accordingly, the vectors derived from AAV only retain the ITRs; the internal coding region is replaced by the desired transgene(s) and regulatory elements (Samulski et al. 1989). To produce recombinant viral particles, a plasmid containing the ITRs flanking the transgene expression cassette is transfected into producer cells in which AAV rep and cap genes as well as necessary helper virus genes are provided either by transfection or by infection.
AAV vectors transduce various types of neurons in the adult rat (McCown et al., 1997, Klein et al., 1998) and monkey (During et al., 1998) as well rodent and human neurons in culture (Du et al., 1996). Human brain slices from epileptic patients could also be transduced by AAV vectors (Freese et al., 1997). AAV vectors were shown to integrate in neurons (Wu et al., 1998).
The international PCT/US95/04587 describes gene delivery to adult CNS using AAV vectors.
However, controllable gene expression is a prerequisite for safe gene therapy or gene immunization in many protocols: for example, erythropoietin level is critical for the treatment of xcex2-thallaseemia.
In models for Parkinson""s disease, the intrastriatal delivery of AAV viral vectors encoding GDNF resulting in long-term overexpression of GDNF effectively protects dopaminergic neurons but also results in side-effects on neighboring normal cells (Kirik et al., 2000). PCT/US94/06734 describes a prokaryotic tetracycline system to produce a genetic switch for achieving control of eukaryotic gene expression. In the native prokaryotic tetracycline system, tetracycline is an effector that induces prokaryotic gene expression by binding to a tetracycline repressor protein. In the absence of tetracycline, the tetracycline repressor binds to a tetracycline operator sequence, which is linked to a promoter and represses transcription. In the presence of tetracycline, the tetracycline repressor binds tetracycline, which binding displaces the repressor from the tetracycline operator sequence , so repression is relieved and transcription can begin.
This tetracycline-controlled activator system is constructed by fusing a tetracycline repressor to a transcription activation domain from a protein that activates transcription in eukaryotic cells. In the absence of tetracycline, the tetracycline-controlled activator (tTa) binds the tetracycline operator sequence which is linked to a promoter and activates transcription. In the presence of tetracycline, the tetracycline-controlled activator binds tetracycline, which binding displaces the activator from the tetracycline operator sequence so activation is ended and transcription is silenced. This is a tetracycline-repressible system.
In a further adaptation, the tetracycline-transactivator is mutated in such a way that it binds the tetracycline operator sequence only when tetracycline binds to the mutant tetracycline transactivator (rtTA). Consequently, in the absence of tetracycline, transcription does not occur. In the presence of tetracycline, transcription can begin. This is a tetracycline-inducible system.
These regulatory systems require that two different expression vectors enter each cell. A first expression vector encodes the tetracycline-controlled activator. A second expression vector encodes the desired transgene under the control of the tetracycline operator sequence linked to a promoter.
However, the probability of transfecting a single cell with two plasmid DNAs is significantly lower than for transfecting that cell with one plasmid DNA. Furthermore, it is important for regulation of expression that the two constructs are present in the optimal ratio.
The U.S. Pat. No. 5,891,718 patent describes a self-accelerating plasmidic system inducible by tetracycline and its analogs, using 2 genes (tTA or rtTA and the gene of interest) expressed from the same tetracycline-inducible/repressible promoter and separated by an xe2x80x9cInternal Ribosome Entry Sitexe2x80x9d (IRES).
This one-plasmid system inducible by tetracycline was shown to be suitable for transient expression in muscle. However, naked DNA is not effective in gene transfer in other organs, for example in the brain. Furthermore, naked DNA does not seem to be effective for long-term gene expression.
In contrast, AAV vectors are effective in long-term gene delivery and expression, in particular in the brain, muscle and liver.
Furthermore a self-accelerating system is based on low level background expression of the tetracycline transactivator and concomitantly of transgene resulting from the basal activity of the minimal CMV promotor (which is part of the tetracycline-responsive element). Therefore, this system is by essence leaky.
Accordingly, the U.S. Pat. No. 5,891,718 patent describes 40-fold induction of transgene in vivo in the muscle. However, background expression in the absence of tetracycline is still detectable in this system.
Bohl et al. (1998) describe a AAV vector expressing erythropoietin (EPO) under a tet-repressible promoter. After injection in the muscle, in the presence of tet EPO is expressed, in the absence of tet EPO is expressed at reduced level but still significantly higher, than the background level. In this construct, the reverse tetracycline transactivator (rtTA) is under the control of a constitutive retroviral promoter and EPO is under the control of tetracycline operator (tetO) sequences linked to a minimal CMV promoter (miniCMV). In this system the expression of the EPO transgene is induced xcx9c10 fold in response to doxycylin.
Haberman et al. (1998) describe a AAV vector expressing gfp under a tet-repressible promoter. This is a single construct with tTA under control of tetO miniCMV and the reporter gene coding for xe2x80x9cgreen fluorescent proteinxe2x80x9d (gfp) under the control a second tetO miniCMV. Thus this is a self-accelerating system After infusion in the brain, in the absence of tet, gfp is expressed; in the presence of tet gfp is expressed at reduced level but still significantly higher than the background level. In this system, the addition of tetracycline results in a xcx9c15-fold reduction of gfp expression.
The presence of a high background level of transgene expression in these two systems could originate from 2 difficulties.
i) Flotte et al. (The Journal of Biol. Chem. 268, 3781-3790, 1993) showed that AAV ITRs have a promoter activity. Thus , in the constructs by Bohl et al. and Haberman et al. tet-independent transcription of gfp can occur from AAV ITRs.
ii) The enhancer elements present in the retroviral LTRs are acting at distance and in both orientations.
Paulus et al. (J. Virol. 70, 62-67, 1996) describe a retroviral vector which proposes a solution for problems nxc2x0i) and ii):
They constructed a LTR-TRE-lacZ-IRES-tTA vector in which: xe2x80x9cpotential cis-regulatory problems in the tetracycline regulation of the phCMV-1 promoter due to proximity to potent Moloney viral enhancer and promoter elements are eliminated by the use of a SIN vector which lacks these elementsxe2x80x9d.
Flotte et al. (ref) showed that AAV ITRs have a promoter activity. Thus , tet-independent transcription of gfp can occur from AAV ITRs.
The present invention aims to provide a new inducible/repressible genetic construct and a vector comprising it, which do not present the drawbacks of the state of the art and which improve the control of the expression of gene(s) of interest, especially in the field of gene therapy and gene immunization.
A preferred aim of the invention is to provide a single self-accelerating AAV viral vector that contains all the elements necessary for a tight regulation on a single construct smaller than 4.7 kb which can be encapsidated using plasmids expressing capaids from AAV-2 or AAV5 and required adenoviral genes.
A further aim of the invention is to provide strong transcription termination insulating the ITRs from the transgenes and consequently avoiding tet-independent transcription.
It is also an aim of the invention to reduce background expression of the transgene due to basal activity of the bidirectional tetracycline-inducible promoter to level undetectable using quantitative methods known by the one skilled in the art (for example: ELISA, fluorimetry, spectrophotometry, etc.).
The present invention is related to an antibiotic inducible/repressible genetic construct (or system) for improving the control of gene therapy and gene immunization and which (by the administration of said antibiotic 5 to a patient) induces the transcription and the expression of one (or more) gene(s) of interest incorporated in said genetic construct by a transfected cell or tissue. According to the invention, said genetic construct 1, controlling the transcription of the gene of interest 2 by said cell 3, comprises a bi-directional antibiotic controlled activator-responsive promoter/operator sequence 4 located between (and controlling the transcription of) the gene of interest 2 (or an insertion site for said gene of interest 2) and a cistron 6, encoding a reverse antibiotic controlled traneactivator 7. In said construct or system the bi-directional antibiotic controller activator responsive promoter/operator sequence 4 is advantageously activated by the transactivator factor 7, encoded by the reverse antibiotic controlled transactivator nucleotide sequence 6 in the presence of said antibiotic 5 (see FIG. 6).
Advantageously, the antibiotic elements used according to the invention is the tetracycline or its analogue (such as doxycyclin) and the reverse antibiotic controller activator is a reverse tetracycline controlled traneactivator (rtTA), whose sequence has been described by Goossen et al., Science, Vol. 278, p. 1766-1769 (1995) and preferably obtained from the Clontech Laboratories catalogue.
Preferably, the bi-directional antibiotic controller activator responsive promoter/operator sequence 4 comprises, located between two mini CMV-promoter, an antibiotic responsive element, preferably a tetracycline responsive element (TRE), consisting of 7 copies of the 42 base pairs of the tetracycline operator sequence, such as the one described by Baron U. et al., Nucleic acid research, Vol. 17, p. 3605-3606 (1995).
Such bi-directional sequence (Pbi-1) is described in the Clontech Laboratories catalogue.
The genetic construct 1 further comprises (preferably bi-directional) terminator sequences such as (SV40 poly-A) poly-adenylation sequences 12 at its both extremities.
Advantageously, the gene construct 1 according to the invention further comprises downstream the antibiotic controlled transactivator sequence 6, another cistron comprising one (or more) gene(s) of interest 2 (or integration site of one (or more) gene(s) of interest 2) and an internal ribosone entry site (IRES) 9 positioned between said gene of interest 2 or integration site of gene of interest and the second nucleotide sequence 6, encoding the reverse antibiotic controlled transactivator 7.
It is meant by a site of integration of a gene of interest 2, a specific genetic sequence (such as polylinker or a genetic sequence comprising a unique restriction enzyme site) allowing the incorporation of a (foreigner) gene of interest 2 incorporation in the genetic construct 1 according to the invention.
Another aspect of the present invention is related to a vector 10 comprising the genetic construct 1 according to the invention. Preferably, said vector is selected from the group consisting of plasmids, viruses, cationic vesicles or a mixture thereof. Advantageously, the vector 10 comprises the genetic construct 1, disposed between two terminal encapsidation, integration and replication viral genetic sequences 11, preferably between two adeno-associated viral (AAV) or adeno-viral ITR-sequences, two retro-virus LTR-sequences or two palindromic sequences of an autonomous parvovirus.
Preferably, the vector 10 according to the invention comprises also between the genetic construct 1 and said terminal sequence 11, two SV40 bi-directional poly-adenylation (Poly-A) sequences 12.
Furthermore, the nucleic acid construct 1 or the vector 10 according to the invention comprises advantageously a sequence encoding an antibiotic silencer sequence 13 located between the viral terminal sequences 10 and the (bi-directional) SV40 poly-adenylation sequences 12.
In the genetic construct 1 according to the invention, the genes of interest 2 are advantageously therapeutic genes, preferably selected from the group consisting of anti-apoptotic sequences (such as bel-2), sequences encoding neurotrophic factors, preferably GDNF, BDNF, NT4 or CNTF, genetic sequences encoding protein such as erythropoietin (EPO), a human growth factor, a tissue granylocyte macrophage colony stimulator factor (GM-csF), tissue plasminogen activator (tPA), coagulation factors (FVIII, FIX), insulin, calcitonyn, thimidine kinase, interleukins (IL-2, IL-6, . . . ), interferons (xcex1, xcex2, xcex3), tumor necrosis factor, genes encoding enzymes involving the detoxification of free radicals (such as superoxide dismutase or the genetic sequence described in the document PCT/BE98/00124, incorporated herein by reference), genetic sequences encoding tumor-specific antigens such as antigens MAGE 1, MAGE 3, etc.).
Another aspect of the present invention is related to a cell (including stem cells) and/or tissue (preferably a mesencephalic tissue or a striatal tissue) transformed by the genetic construct or the vector according to the invention, preferably a cell which could be used also as a cellular vector or a vaccine for genetic and/or cellular therapy and immunization. Preferably, said genetic therapy using the nucleic acid construct or vector according to the invention allows a transformation of cells obtained from foetal-nervous tissues which are thereafter used for the treatment of neurodigenerative diseases, especially for the treatment of Parkinson disease, Alzheimer or Huntington disease.
Another aspect of the present invention is related also to a method for improving the survival of graft of foetal-nervous tissue by inducing a genetic modification in the cell(s) of said tissue with a viral vector (preferably a AAV-vector, more preferably the vector according to the invention) encoding a neurotrophic factor for improving the treatment of neuro-degenerative diseases, especially Parkinson disease (using for example GDNF) and Huntington disease (using for example CNTF).
More preferably, said neurotrophic factor is the one described above.